Radix Astragali and Its Representative Extracts for Diabetic Nephropathy: Efficacy and Molecular Mechanism

Diabetic nephropathy (DN) is a common microvascular complication of diabetes mellitus (DM). Radix Astragali (RA), a frequently used Chinese herbal medicine in the Leguminosae family, Astragalus genus, with its extracts, has been proven to be effective in DN treatment both in clinical practice and experimental studies. RA and its extracts can reduce proteinuria and improve renal function. They can improve histopathology changes including thickening of the glomerular basement membrane, mesangial cell proliferation, and injury of endothelial cells, podocytes, and renal tubule cells. The mechanisms mainly benefited from antioxidative stress which involves Nrf2/ARE signaling and the PPARγ-Klotho-FoxO1 axis; antiendoplasmic reticulum stress which involves PERK-ATF4-CHOP, PERK/eIF2α, and IRE1/XBP1 pathways; regulating autophagy which involves SIRT1/NF-κB signaling and AMPK signaling; anti-inflammation which involves IL33/ST2 and NF-κB signaling; and antifibrosis which involves TGF-β1/Smads, MAPK (ERK), p38/MAPK, JNK/MAPK, Wnt/β-catenin, and PI3K/AKT/mTOR signaling pathways. This review focuses on the clinical efficacy and the pharmacological mechanism of RA and its representative extracts on DN, and we further document the traditional uses of RA and probe into the TCM theoretical basis for its application in DN.


Introduction
Diabetic nephropathy (DN), also known as diabetic glomerulosclerosis, is a series of renal pathological changes resulting from diabetes mellitus (DM), including glomerular basement membrane (GBM) thickening, mesangial expansion, extracellular matrix (ECM) deposition (mainly mesangial), and tubular atrophy, leading to renal interstitial fibrosis and glomerulosclerosis [1][2][3].As one of the most prevalent diabetic microvascular complications, DN has become the leading cause of end-stage kidney disease (ESRD) [3].It is clear that mechanisms like oxidative stress, endoplasmic reticulum (ER) stress, inflammation, and autophagy impairment are involved in the pathological changes aforementioned [1].However, the treatment of DN is still mainly limited to the control of blood glucose and blood pressure so far [4].
For DN treatment, Chinese herbal medicine (CHM) shows the modulative function of metabolism, inflammation, oxidative stress, ER stress, and fibrosis and has been proven useful in clinical practice for glycosylated hemoglobin (HbA1c), fasting blood glucose (FBG), urinary albumin excretion rate (UAER), serum creatinine (Scr), 24-h urinary protein (24 h UP), and estimated glomerular filtration rate (eGFR) [5].Obviously, CHM is a promising therapeutic tool in DN treatment.
Radix Astragali (RA) is one of the most widely used herbs for the traditional Chinese medicine (TCM) treatment of DN, which is the dry root of Astragalus mongholicus Bunge or Astragalus membranaceus (Fisch.)Bunge [6].It was first recorded in the book Prescriptions of Fifty-Two Diseases, written in the Warring States period (475-221 BC).Its main therapeutic effect is tonifying qi.The main pharmacological components of RA include polysaccharides, saponins, and flavonoids [7].Among these components, astragalosides take the highest proportion of triterpene saponin content in RA [7].As the marking compound of RA, astragaloside IV (AS-IV) has been documented with the functions of neuroprotection, liver protection, hematopoietic system protection, antivirus, antibacteria, immunity enhancement, antitumor, and antidiabetes and its complications [8,9].In addition, as the highest content component of RA, astragalus polysaccharide (APS) has also been widely studied, including the efficacy of immune regulation, antiaging, antitumor, reducing blood glucose and blood lipid, antifibrosis, antibacterial, antivirus, and radiation protection [10].Flavonoid compounds in RA are mostly calycosin-7-O-β-D-glucoside, calycosin, ononin, and formononetin.Calycosin-7-O-β-Dglucoside and calycosin are the representative constituents of RA and have been proven with antitumor, neuroprotective, antidiabetic, antiosteoporosis, liver protection, and cardiovascular system protection effects [11,12].With the extensive efficacy of these components above, recent research has provided insight into the value of RA in the treatment of tumors, coronary heart disease, diabetes, nephritis, and so on [6,9,10,12].

Therapeutic Effects of RA on DN
2.1.Clinical Efficacies.For DN, the therapeutic effect of RA has been proved by many clinical trials.A meta-analysis of 21 RCTs and 4 CCTs with 1804 subjects assessed the efficacy of intravenous drip of RA injection in treating Stages III-IV (Mogensen) DN.Results showed that RA injection contributed to a better effect in decreasing blood urea nitrogen (BUN), Scr, creatinine clearance rate (Ccr), and urinal protein, and increasing serum albumin (ALB) level compared with the control group (ACEi/ARB) [13].The updated meta-analysis of 66 RCTs with 4785 patients (no restriction on the stage of renal function) including all forms of RA preparation (including tablet, granule, decoction, extract liquid, and injections) showed that adjunctive use of RA preparations on conventional therapies of ACEi/ARB could reduce albuminuria, proteinuria, and Scr in DN patients [14].A retrospective cohort study among 6648 predialysis DN patients found RA users with lower all-cause mortality compared with other CHM users after a 5-year follow-up [15].Here, we summarize the study design, main conclusions, and safety outcomes of the clinical research on RA in treating DN in the past 10 years (see Table 1).The preparation mostly used is RA injection (from 20 to 50 mL qd).RA tablets (2.2 g, bid), granules (from 4 to 15 g, qd or bid), and APS injection (250 mg, qd) are also common preparations and dosages.These indicate that the optimal dose for RA injections and oral preparations has not been fully vali-dated, and whether the injection or the oral preparations can give play to higher effects still needs investigation.The frequent adverse effects (AEs) in these studies include gastrointestinal reactions, liver function damage, hyperkalemia, cough, dizziness, and headache.Parts of these AEs are common in ACEI/ARBs treatment, and no statistical difference was reported in the incidences of AEs between the treatment group and the control group.Therefore, it is not yet clear about the AEs of RA preparations.Besides, 31 out of these 50 researches did not report safety outcomes, suggesting insufficient attention to AEs of TCM.Anyhow, RA preparations exert a therapeutic effect on DN by reducing urinary protein and decreasing BUN, Scr, and Ccr.These finally result in a reduction of endpoint events, indicating the supplementary treatment value of RA for DN.
2.2.Histological Effects.When injuries occur to renal cells, the responses vary from hypertrophy, proliferation, activation, and transformation to necrosis and apoptosis.These cells can secrete a variety of cytokines and inflammatory mediators and produce varying amounts of ECM, resulting in glomerulosclerosis and tubulointerstitial fibrosis; this is how renal function is damaged [66].If we can balance cell proliferation and apoptosis, control cell activation, and phenotypic transformation, it will greatly suppress the histopathology changes of renal tissue and increase the possibility of curing and delaying nephropathy.Therefore, it is of great significance to study the pathological effects in the treatment of disease and injury.Based on the current studies, RA has shown protective effects on specific renal intrinsic cells and emerged as a promising treatment through the modulation of multiple pathogeneses in DN progress, especially in renal fibrosis (Figure 1).Detailed effects include reducing ECM deposition; alleviating GBM and TBM thickening; ameliorating mesangial cell (MC) proliferation, hypertrophy, and contractile dysfunction; alleviating mesangial hyperplasia; inhibiting glomerular endothelial cell (GEC) apoptosis; restoring increased GEC permeability and capillary loop diameter and decreased podocyte autophagy, podocin, and nephrin expression; suppressing podocyte apoptosis, detachment, and foot process effacement; decreasing foot process width; reversing promoted epithelial-mesenchymal transition (EMT) in podocytes and renal tubular epithelial cells (TECs) (RTEC); inhibiting TECs' edema and apoptosis; and so on.

Mechanisms of RA and Its Representative
Extracts on DN RA and its representative extracts exhibit the renal protective effect in DN through multiple mechanisms independent of antihyperglycemic.[67].Hyperglycemia promotes the generation of advanced glycation end products (AGEs) in the cells, which trigger mitochondria to release excess reactive oxygen species (ROS), resulting in renal inflammation, renal cell injury, and fibrosis [5,68].The astragalosides in RA, mostly AS-IV, exhibit significant antioxidant effects.In diabetic db/db mice, AS-IV ameliorated podocyte injury by enhancing klotho expression and inhibiting oxidative stress through the PPARγ-Klotho-FoxO1 axis [69].In STZ-induced hyperinsulinemia rats, AS-I could decrease the level of AGEs [70] and suppress the activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (Nox4) expression and the phosphorylation of ERK1/2.These actions further reduce malondialdehyde (MDA) and increase activities of antioxidant enzymes such as glutathione (GSH) peroxidase (GSH-Px), superoxide dismutase (SOD) [71,72], and catalase (CAT) [73] and increase total antioxidative capability (T-AOC) [70], thus inhibiting oxidative stress.
In vitro results were consistent with those from animal studies.AS-IV reduced the content of ROS [73], restored Klotho expression, enhanced forkhead box transcription factor O1 (FoxO1)-dependent antioxidant activity, and upregulated Nrf2-ARE/TFAM signaling to resist oxidative stress damage, protecting HG-exposed podocytes from apoptosis [69,72].AS-IV also inhibited MC proliferation and glomerular contractile dysfunction caused by HG culture.These effects were achieved through the NADPH oxidase/ROS/ Akt/NF-κB pathway [74,75].The inhibitory effect of AS-IV on oxidative stress can also protect renal tubular cells.Qi et al. [76] proved that AS-IV could attenuate glycosylated albumin (GA)-induced amplification of ROS, inhibit NADPH oxidase activity, and elevate the level of SOD units in the NRK-52E cell line.In HG-exposed HK-2 cells, AS-IV could regulate the Wnt/β-catenin pathway and Nrf2/ARE signaling pathway, an endogenous antioxidant stress pathway; further enhance the activities of SOD, GSH-Px, and CAT; and decrease the generation of MDA and ROS [77,78].Another study on HG-induced HK-2 cells also found that AS-IV could alleviate palmitic acid (PA)-induced cell apoptosis by decreasing ROS production and increasing P-Nrf2 levels [79].These antioxidant processes lead to the prevention of cell apoptosis, which might be associated with the reduction of apoptotic proteins like Bax, cleaved-caspase-3, cleaved-caspase-9, and Bcl-2 [73,77,79].

Inhibition of ER Stress.
The main physiological function of the ER is to synthesize proteins.Pathophysiological stress conditions may interfere with normal protein folding and cause accumulation and aggregation of misfolded and unfolded proteins (UPR), inducing cellular toxicity.ER stress occurs when excess UPR accumulates beyond the load that the cell can deal with [85].HG condition leads to altered protein glycosylation, which can interfere with protein folding, causing the accumulation of misfolded and UPR in the ER and contributing to ER stress, which could be cytotoxic after prolonged processes [85,86].
In HFD and STZ-induced DN rats, intragastric AS-IV could alleviate RTEC apoptosis by downregulating the expression of ER stress-related proteins like p-PERK, ATF4, and CHOP and restoring the balance of Bax and Bcl-2 expression [87].In STZ-induced DN rats, AS-IV could also alleviate ER stress-induced podocyte apoptosis by suppressing the PERK-ATF4-CHOP pathway [88]; inhibiting the expression of ORP150 and GRP78; and downregulating the phosphorylation of PERK, eIF2α, and JNK [89].In db/ db mice and podocytes under HG or PA stimulation, AS-IV ameliorated podocyte apoptosis by attenuating ER stress by restoration of SERCA2 expression and activation [90,91].Overall, AS-IV exerts its anti-ER stress effect by blocking the PERK-ATF4-CHOP, PERK/eIF2α, and IRE1/JNK pathways; upregulating SERCA2 expression; and downregulating ORP150 and GRP78 expression.

Control of Regulated Cell Death (RCD).
Physiologically, RCD clears unwanted cells, but in some pathological situations, disproportionate responses can contribute to the detrimental loss of kidney cells [92].Based on molecular and essential aspects of the cell death process, the RCD process can be categorized into different modalities such as intrinsic apoptosis, extrinsic apoptosis, MPT-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, and immunogenic cell death.
Pyroptosis is triggered by perturbations of extracellular or intracellular homeostasis related to innate immunity manifesting with a peculiar form of chromatin condensation as well as cellular swelling culminating with plasma membrane permeabilization [93].Pyroptosis generally relies on the activation of one or more pyroptotic caspases (caspase-1, caspase-4, caspase-5, and caspase-11) and is always associated with the activation and secretion of the two prominent proinflammatory cytokines, interleukin-1β (IL-1β) and IL-18 [94].In the db/db mouse model, AS-IV inhibited the expression levels of NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome, apoptosis-associated speck-like protein (ASC), caspase-1, and IL-1β in the renal cortex and reduced the serum levels of TNF-α and monocyte chemoattractant protein-1 (MCP-1), thus protecting podocytes from pyroptosis accompanied by the upregulation of the podocyte markers podocin and synaptopodin [95,96].In STZ-induced rats, oral treatment with calycosin decreased Scr, BUN, and proteinuria levels; escalated urine Ccr; and alleviated interstitial collagen deposition and GBM thickening.These alternations were accompanied by decreased levels of serum LDH and IL-1β, renal NF-κBp65, pyroptotic-related proteins (TXNIP and NLRP3), and MDA and elevated IL-10, TAC, and Nrf2 expression, indicating that the renal-protective effect of calycosin might be associated with the modulation of oxidative stress NLRP3 and TXNIP-mediated pyroptotic signaling [84].
Ferroptosis is a form of RCD characterized by the irondependent accumulation of lipid hydroperoxides.It could be induced by the accumulation of glutamate, iron, or polyunsaturated fatty acid phospholipids, or by depletion of endogenous inhibitors of ferroptosis, such as reduced GSH, NADPH, Phospholipid Peroxidase Glutathione Peroxidase 4 (GPX4), or vitamin E [97].Calycosin has been proven to be able to regulate ferroptosis in diabetic kidney disease through in vivo and in vitro studies.In db/db mice, it decreased BUN and Cr, alleviated tubular dilatation and collagen deposition, and reversed the loss of the proximal tubule brush border.In HG-treated HK-2 cells, it increased cell viability.These were achieved by the upregulation of GPX4, inhibition of lipid ROS production, and nuclear receptor coactivator 4 (NCOA4) expression, which are all correlated with ferroptosis [98].
Autophagy, a process of degrading damaged proteins and macromolecules, is a cellular protective mechanism.The autophagy levels of renal cells (including podocytes, proximal renal tubules, MCs, and endothelial cells) are downregulated in hyperglycemia conditions, which can promote the development of DN [99].AS-IV can restore the autophagy of podocytes by inhibiting AMPKα activation, as proved by the upregulation of autophagy-related proteins LC3A/B, Beclin-1, and autophagy-related protein 12 (Atg12) and downregulation of autophagy-related protein p62 (p62) [90].In MCs, AS-IV can also regulate Beclin-1 and LC3 II through SIRT1/NF-κB signaling, subsequently suppressing MC proliferation [100].
3.4.Anti-Inflammatory Effect.Diabetes can be regarded as chronic low-grade inflammation or a preinflammatory state.Inflammatory cytokines, such as TNF-α, IL-1β, and IL-6, activate a variety of signal pathways, resulting in insulin resistance and decreased insulin secretion, and promote the development of diabetes [101].

Inhibition of Renal Fibrosis.
Renal fibrosis is characterized by tubule atrophy, interstitial chronic inflammation and fibrogenesis, glomerulosclerosis, and vascular rarefaction [106].Abnormal activation of several cell growth factors such as transforming growth factor beta (TGF-β), connective tissue growth factor (CTGF), angiotensin II (Ang II), hepatocyte growth factor (HGF), and their receptors are the mediators of renal fibrosis [107,108].
In the human glomerulus, known cell types include GECs, podocytes, MCs, and parietal epithelial cells.Podocyte injury results in their detachment and loss, which is one of the major determinants of glomerular injury and glomerulosclerosis.The manner in which endothelial cells contribute to glomerulosclerosis is to regenerate and produce GBM, causing the thickening of GBM.Similarly, the pathological changes of MCs and parietal epithelial cells during fibrosis progress are to regenerate, proliferate, and produce ECM, which also potentially leads to glomerulosclerosis [106,109].
As for the renal tubule, in the DN process, infiltrating immune cells, glucosuria, and albuminuria can trigger the secretion of proinflammatory and profibrotic mediators in proximal TECs, resulting in interstitial inflammation and fibrosis [110].EMT is one of the pathognomonic pathological processes of interstitial fibrosis, which manifests as the loss of the original phenotype of TECs, the destruction of the renal tubular basement membrane, the shift of TECs from the renal tubules to the interstitium through the damaged basement membrane, and the transformation of TECs into myofibroblasts expressing α-smooth muscle actin (α-SMA) [108].Another typical pathological change of interstitial fibrosis is ECM accumulation, which represents the excess accumulation of matrix proteins including Types Ι, III, V, VI, VII, and XV collagen, as well as the adhesive glycoprotein fibronectin (FN), which results in the expansion of the space between the tubular basement membrane and peritubular capillaries [111].
Additionally, in vitro and in vivo studies demonstrated that calycosin treatment could inhibit renal ECM deposition and modulate fibrotic processes [80,82].
3.6.Other Mechanisms Against DN.Hyperglycemia-induced GEC injury manifests as cell dysfunction, even apoptosis.Endothelial dysfunction in diabetes is associated with impaired abilities of nitric oxide (NO) production and eNOS activation [122].AS-IV has been proven to be able to promote the synthesis of NO, enhancing eNOS phosphorylation and activity, thereby reversing HG-treated human renal GEC permeability and apoptosis [123].
The pathological changes of podocytes in DN include hypertrophy, dedifferentiation (EMT), impaired autophagy, podocyte detachment, effacement, and apoptosis [68].α3β1 integrin acts as an anchor to connect the podocyte with the GBM.AS-IV can increase the expression of α3β1 9 Journal of Diabetes Research integrin and decrease the expression of integrin-linked kinase (ILK), subsequently ameliorate podocyte adhesion dysfunction, inhibit podocyte detachment [124], and increase podocyte density [125].TRPC6, one of the Ca 2+ ion channels expressed in podocytes, is involved in NFATdependent cell apoptosis.AS-IV can prevent HG-exposed podocyte apoptosis via downregulation of TRPC6, NFAT2, and Bax expression [126].Apoptosis of podocytes is also associated with TRAF5-mediated NF-κB activation [127].AS-IV increases the expression of miR-378 and elevates the TUG1 level, both of which can attenuate the TRAF5 level, thus suppressing podocyte apoptosis in DN rats and HG-treated MPC5 cells [128,129].
Adiponectin is an adipose-derived hormone.It has proven to be beneficial in DN by means of a comprehensive effect of antioxidation, anti-inflammatory, modulating RCD, anti-ER stress, antifibrosis, and improving endothelial dysfunction.However, during the progress of glucose and lipid metabolism disorders, the secretion of adiponectin is downregulated [130][131][132].In a db/db mouse model, HG-treated human GECs, and murine podocytes, AdipoRon, a synthetic adiponectin receptor agonist, showed the ability to activate intrarenal AdipoR1 and AdipoR2, followed by activating the CaMKKb/phosphorylated Ser431LKB1/phosphorylated Thr172AMPK/PPARα pathway, thus decreasing oxidative stress and apoptosis and improving endothelial dysfunction [133].Two other compounds derived from RA (astragaloside II and isoastragaloside I) can activate AMPK via the increase of adiponectin secretion in db/db mice [134].Therefore, increasing the secretion of adiponectin may be an important way for RA to treat DN.
Another study on db/db mice demonstrated that AS-IV reduced glomerular mesangial matrix, proximal tubular area, and urinary NAG (a marker of proximal tubular injury) excretion by modulating the mitochondrial quality control network, as evidenced by the downregulation of the levels of mitochondrial fission-associated protein including Drp-1, Fis-1, and MFF and mitophagy-associated protein including PTEN-induced putative kinase 1 (PINK1), Parkin, p-Parkin (Ser 65), and LC-3II [135].
The pharmacological effects and targets of RA and its representative extracts on DN are summarized in Table 2 and Figure 2.

Traditional Views and the Link With Pharmacological Properties of RA
In TCM theory, qi is the source of energy, constituting the human body and sustaining its life activity, exerting a positive regulatory effect on various life activities.The qitonifying efficacy of RA has long been recognized, since the Shennong Materia Medica Sutra, which was completed in the Eastern Han Dynasty (25-220 AD), has described RA as "tonifying deficiency, curing illness of children, treating exogenous diseases, and cutaneous infection."In later generations, based on the qi-tonifying efficacy, the indication of RA extended to various syndromes involving the pathogenesis of qi-deficiency, including strengthening the body, consolidating the defensive function of the body (for treating fatigue, spontaneous sweating, loss of appetite, etc.), facilitating dispelling pathogenic qi (for treating exogenous diseases and cutaneous infection), propelling blood (for treating numbness and pain in limbs) and body fluid (for treating edema), and preventing the leakage of essences (for treating diarrhea, polyuria, proteinuria, and hematuria).
Based on these traditional uses, RA has been much more indepth applied.Its anti-inflammatory, antioxidation, anticancer, immunomodulation, and antiviral mechanisms can be related to the positive regulation of qi on the human body, thereby exerting the effects of antiaging, antidiabetes, heart protection, renal protection, liver protection, and neuroprotection [7,136,137].TCM regards heat impairing qi and yin as the basic pathogenesis of DM, and the qi-deficiency is persistent in the course of DN from the beginning to the end stage.With the development of DN, qi-deficiency gradually becomes more severe, damaging other physiological functions, such as propelling body fluid, preventing the leakage of urine protein, and excreting toxins.Modern studies support that qideficiency syndrome is closely related to immune dysfunction, oxidative stress, inflammation, abnormalities in energy metabolism, and so on [138].In the qi-deficiency animal models, the contents of eNOS and NO and SOD activity decreased, and the content of MDA and inflammatory factors like IL-1β, IFN-γ, IL-6, IL-8, and TNF-α increased [138].These findings coincide with the changes in the progression of DN.
The components of another typical CHM with the primary efficacy of qi-tonifying, Panax ginseng C.A. Mey.(Renshen), have been proven to show similar pharmacological properties to the RA extracts, such as regulating oxidative stress, inflammation, autophagy, and apoptosis [139] and increasing NO synthesis [140].Some TCM prescriptions or herbal pairs with qi-tonifying as the core function also suggest the potential mechanism of the qi-tonifying effect.Powder for restoring pulse beating (Shengmai Powder), decoction of four mild drugs (Sijunzi Decoction), and other TCM prescriptions for qi-tonifying, as well as commonly used herbal pairs such as ginseng and RA, have been proven to be able to regulate Akt signaling in diabeticrelated cognitive decline models and reverse memory deficits in diabetic rats [141].In the ischemic heart disease model, these prescriptions or herbal pairs can regulate AMPK, IL-6 [142], and TNF signaling [143] and upregulate Nrf2 expression, inhibiting JNK phosphorylation and cardiomyocyte apoptosis [144].In Alzheimer's disease, they can regulate IL-1β and glycogen synthase kinase-3β (GSK-3β) signaling and mitigate the oxidative damage of nerve cells [145].In chronic kidney disease (CKD), they can enhance mitochondrial energy generation and antioxidant status; eliminate free radicals; protect renal cells; improve renal microcirculation; and reduce Scr, BUN, and urinary protein [146].Based on the above findings, the mechanisms and effects of qi-tonifying can correspond to the mechanisms and effects of RA and its extracts for treating DN.
By virtue of its superior qi-tonifying efficacy, RA positively affects DN from the perspective of both TCM and modern medicine.The effectiveness of medication based 10 Journal of Diabetes Research

AEs and Toxicity of RA
The AEs and toxicity of RA have been studied.In most in vitro studies, the concentrations of AS-IV were under 100 μmol/L, and no evidence of cytotoxicity was shown, while AS-IV 200 μmol/L could decrease cell viability [77,79].When treated with calycosin at doses above 10 μmol/L, a decrease in cell viability was observed [105].As for in vivo experiments, feeding experiments using APS and total flavonoids showed no obvious AEs, and toxicity tests of RA total glucoside, APS, and astragalus saponins revealed that the safety dosage ranges of these components are more than 35 times the dose of humans [136].RA and its extracts demonstrate the renal protective effect in DN through the inhibition of fibrosis, oxidative stress, endoplasmic reticulum stress, inflammation, cell apoptosis, endothelial dysfunction, and promotion of autophagy, podocyte adhesion, and so on.These actions are achieved by the regulation of Bcl-2 and caspase families and signaling pathways including TGF-β1/Smads, Nrf2/ARE, MAPK/ERK, p38/MAPK, JNK/MAPK, Wnt/β-catenin, PI3K/AKT/mTOR, AMPK, NF-κB, PERK-ATF4-CHOP, IREα/XBP1, and PPARγ-Klotho-FoxO1.17 Journal of Diabetes Research Some clinical trials also reported some AEs of RA.In a meta-analysis of clinical studies using RA preparations to treat DN, 20 out of 66 studies reported safety outcomes [14].Among the 376 participants in the five studies that reported AEs, there were 18 cases of dry cough (16 cases were treated with benazepril, and symptoms relieved without treatment within 2 weeks), 15 cases of Scr elevated more than 30% from baseline (Scr went down with treatment later in 13 cases), 13 cases of dizziness, three cases of angioedema, and a case of hyperkalemia.Since all the studies took ACEi/ ARB as the initial therapy, which may lead to all the AEs aforementioned, and no statistical difference in the incidences of each AE was found between the treatment group and control group, the AEs of RA itself seem insignificant.However, in consideration of the data of these five studies, the facticity of the safety outcomes of the other 15 studies which reported no AEs needs to be questioned.According to the results summarized in Table 1, the AEs of RA preparations have received more attention in recent years.These outcomes indicate that serious AEs have not been found in the treatment of DN with RA preparations and, at the same time, throw light on inadequate attention to the safety evaluation of RA preparations in clinical studies.In addition, there is currently insufficient clinical evidence to evaluate the risks and benefits of the use of RA preparations in patients with ESRD.More convincing evidence is still needed.

Discussion and Prospect
The current treatment strategy for DN is still insufficient.In addition to glycemic control, a few drugs, like SGLT2 inhibitors, glucagon-like peptide 1 receptor agonists (GLP-1 RAs), ACEI/ARBs, and finerenone, have been proven to be protective of renal function [147], while side effects are not rare in the application of these drugs, for example, risk of genital infection and diabetic ketoacidosis (DKA) for SGLT2 inhibitors [148]; gastrointestinal events and risk of thyroid C-cell tumors and injection site reactions for GLP-1 RAs [149]; cough, functional renal insufficiency, hyperkalemia, angioedema, and so on for ACEI/ARBs [150]; and hyperkalemia for finerenone [4,151].Much less, renal insufficiency itself is the reason that restricts the use of many drugs.Despite the fact that all these approaches can only slow down but not halt the progression of DN, herbal medicines with composite compounds exhibit efficacy in a multitarget, multipathway manner compared with these chemicals and should be given full focus.
Among the aforementioned extracts, the pharmacological effects of AS-IV are achieved through the mechanisms of antioxidant, anti-inflammatory, anti-ER stress, antiapoptosis, antipyroptosis, antifibrosis, and immune regulation, improving endothelial dysfunction, promoting peripheral nerve regeneration, and limiting lipid deposition [8].APS shows the abilities of immune regulation, anti-inflammation, antioxidant, and anti-ER stress, promoting endothelial cell proliferation, inhibiting liver glycosylation, inhibiting islet β cell apoptosis, and increasing insulin sensitivity in peripheral tissues [10,152].Calycosin exhibits effects of anti-inflammatory, antioxidant, antipyroptosis, and antiferroptosis, promoting osteogenesis, endothelial cell proliferation, and vascular dilation [11,12].The pharmacological mechanisms of these components, which are widely studied, partially overlap, including anti-inflammatory and antioxidation and improving vascular endothelial function.In addition, AS-IV and calycosin have been proven to be able to antipyroptosis and antifibrosis.AS-IV and APS can both exert anti-ER stress, antiapoptosis, antibacterial, antiviral, and immune-regulation effects.The differences among the pharmacological effects of each component may provide new perspectives for drug development for different diseases or pathological processes.Among its representative components, AS-IV, as the constituent for quality evaluation in the Chinese Pharmacopeia, gains the most interest from researchers in the DN field, while APS, calycosin-7-O-β-Dglucoside, calycosin, and other components are relatively less investigated.Based on its comprehensive effects of antioxidation, anti-inflammatory, modulating regulated cell death, anti-ER stress, antifibrosis, and improving endothelial dysfunction, AS-IV could be priorly considered to be developed in the treatment of DN.However, the therapeutic effects for other diseases of APS calycosin-7-O-β-D-glucoside and calycosin also suggest their potential pharmacological mechanisms on DN, and more evidence remains to be explored to provide new strategies for the treatment of DN in the future.
Except for these representative components, network pharmacology studies have found many other flavonoids in RA with multitudinous targets against DN, including quercetin, formononetin, kaempferol, 7-Omethylisomucronulatol, and isorhamnetin [153].Kaempferol acts its renal protective effects through the anti-inflammation, antioxidation, and mechanisms [154][155][156][157].The pharmacological effects of quercetin are similar to kaempferol, which acts on endothelium, TECs, MCs, and podocytes to alleviate DN [158][159][160][161][162].These protective effects are reflected in improvements in the renal index, urine protein, uric acid, urine ALB, and Scr levels [163].Formononetin can also alleviate oxidative stress, restore mitochondrion function, and inhibit renal fibrosis in DN models [164][165][166][167]. Isorhamnetin, as an anti-inflammatory and antioxidation agent, shows a definite antidiabetic effect and the ability to modulate autophagy in renal tissues [168,169].These components share common pharmacological mechanisms with the representative components of RA.Together, the multiple components of RA act in coordination with each other and contribute to the treatment of DN.

Conclusion
Based the present findings, RA and its extracts have a multiscale act on various mechanisms of DN as well as in histopathological changes in most parts of the nephron.Besides, these components show definite protection effects on renal function.Specific mechanisms include inhibition of oxidative stress, inhibition of ER stress, modulation of regulated cell death, anti-inflammation, and inhibition of renal fibrosis.Although many studies have elucidated the

Figure 1 :
Figure1: Main pathological effects of RA against DN.The renal protective effect of RA targets multiple parts of the nephron, including the glomerular basement membrane, mesangium (both mesangial cells and mesangial matrix), endothelium, podocytes, and tubule.Note: Symbols "↑" and "↓" represent increase and decrease, respectively.

-MEK1/ 2 - 2 :
R -K lo th o -F o x O 1 a x Main molecular mechanisms of RA against DN.
3.1.Inhibition of Oxidative Stress.When a free radical, independent chemical species with one or more unpaired electrons, attacks a nonradical molecule, a secondary radical molecule comes into being.The chain reaction of primary and secondary radicals leads to oxidative damage of tissues, while antioxidants can block the initial production of free radicals and the secondary production of toxic metabolites.

Table 1 : Continued. Ref. Baseline renal function or DN stage Participants (T/C) Average age (T/C) Average history of DM (T/C) RA preparations, dose, and frequency Basic therapeutics, dose, and frequency Treatment duration Treatment effects of RA Incidence of adverse effects (T/C) Adverse effects of treatment group
6ALB, microalbumin; Scr, serum creatinine; UACR, urinary albumin to creatinine ratio; UAER, urinary albumin excretion rate.6Journal of Diabetes Research When excessive oxidant compounds and insufficient antioxidants lead to an imbalanced redox state, this is what we call oxidative stress

Table 2 :
Prevention mechanism of RA on diabetic nephropathy.
18 Journal of Diabetes Research therapeutic effect of RA on DN through histopathological changes, clinically, biopsy is not the prior choice of DN patients.The clinical efficacy evaluation still relies on the detection of biochemical indexes.The histopathological examination reflects structure, while biochemical index detection focuses on function, which is what truly matters for patients.The scarcity and low quality of clinical trials failed to provide sufficient evidence to prove how much RA can improve renal function and how safe the different types of RA preparations are, which hiders the usage of RA in clinical practice.It is worth noting that researchers have developed mediums that can encapsulate active extracts of herbal medicine including RA, enhancing the solubility and sustained release, making it possible to promote stable and highly bioavailability herbal ingredient preparations to clinical practice [83, 170, 171].Further, strictly designed large-scale, long-term follow-up trials are warranted to provide definitive evidence for the clinical efficacy of RA and its extracts.Still, these findings pave the way for the future development of RA and its extracts as potential therapeutic preparations in the management of DN.